Anti ifnγ capture antibody

Pan02 cells are MHCI negative (data not shown), and were therefore IFNγ-treated to upregulate MHCI (20 ng/mL, 24h), before mitomycin C treatment (50 μg/mL, 20min) and extensive washing. 8.3 × 10³ Pan02 cells were co-cultured with 8.3 × 10⁴ disaggregated splenocytes from Pan02 tumour-bearing mice (day 29 post-implantation) in RPMI (Life technologies) with 10 % fetal bovine serum (SAFC Biosciences) and 10 U/mL IL-2 for 96 hours in Multiscreen_HTS IP plates (Millipore) coated with anti-IFNγ capture antibody (eBiosciences). The number of IFNγ-producing T cells was then quantified by ELISPOT assay according to manufacturer's instructions (eBiosciences).

Lung cells were isolated using Liberase^TM TM (Roche, Switzerland) and spleen and lymph nodes cells were isolated by mechanical disaggregation as described previously (Grover et al., 2009 (link)). For post-vaccination ELISpot, inguinal, axillary, and brachial lymph nodes were used. For post-infection ELISpot, tracheal bronchial, axillary, brachial, and inguinal lymph nodes were employed. Cells (2 × 10⁵) were then cultured in plates coated with anti-IFN-γ capture antibody according to the manufacturer’s protocol (eBiosciences, San Diego, CA, United States) for 24 h at 37°C, 5% CO₂ in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO, United States), 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 mM L-glutamine (Sigma, St. Louis, MO, United States) in the presence of M. tuberculosis H37Rv-derived culture filtrate protein (BEI Resources, Manassas, VA, United States). Controls included cells incubated in media alone. Spot forming units (SFU) were determined according to the manufacturer’s protocol and the plates were then analyzed by quantifying the number of spots produced by cytokine producing cells using the Series 5 UV-Immunospot Analyzer (C.T.L., Shaker Heights, OH, United States). Analysis of T-cell activation was also performed on lung, spleen, and lymph node cells at day 180 post-infection.